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Human CD4⁺CD25⁺ Regulatory T Lymphocytes Inhibit Lipopolysaccharide-Induced Monocyte Survival through a Fas/Fas Ligand-Dependent Mechanism¹

Fabienne Venet^*, Alexandre Pachot, Anne-Lise Debard, Julien Bohe, Jacques Bienvenu, Alain Lepape, William S. Powell^¶ and Guillaume Monneret^2,*

^* Immunology Laboratory, Hôpital Neurologique, Hospices Civils de Lyon, Lyon, France; Joint Unit bioMérieux, Hôpital Edouard Herriot, Hospices Civils de Lyon, Lyon, France; Lyon-Sud University Hospital, Immunology Laboratory, Hospices Civils de Lyon, Pierre-Bénite, France; Intensive Care Units, Lyon-Sud University Hospital, Hospices Civils de Lyon, Pierre-Bénite, France; and ^¶ Meakins-Christie Laboratories, McGill University, Montreal, Canada

	Abstract

Top Abstract Introduction Patients, Materials and Methods Results Discussion Disclosures References

 
Although it is known that septic shock induces immunosuppression, the mechanism for this phenomenon is not well understood. Monocytes play a central role in septic shock pathophysiology, which is also characterized by an increased proportion of natural regulatory T (Treg) cells. We therefore investigated whether Treg could be involved in the decreased monocyte expression of CD14 and HLA-DR observed during septic shock. We demonstrated that human Treg inhibit LPS-induced retention of monocyte CD14. Because loss of CD14 is a hallmark of monocyte apoptosis, this suggests that Treg inhibit monocyte survival. This effect was largely mediated through the release of a soluble mediator that was not identical with either IL-10 or IL-4. The Fas/FasL pathway participated in the effect as it was blocked by anti-FasL Abs and reproduced by Fas agonist and recombinant soluble FasL. Furthermore, expression of FasL was much higher on Treg than on their CD25^– counterparts. Collectively, these results indicate that Treg act on monocytes by inhibiting their LPS-induced survival through a proapoptotic mechanism involving the Fas/FasL pathway. This may be an important mechanism for septic shock-induced immunosuppression and may offer new perspectives for the treatment of this deadly disease.

	Introduction

Top Abstract Introduction Patients, Materials and Methods Results Discussion Disclosures References

 
Despite the great advances in modern medicine, septic shock remains a major cause of death in intensive care units, with a mortality rate that is regularly reported as high as 40–50% (1, 2). It is now agreed that septic shock deeply perturbs immune homeostasis by inducing an initial intense systemic inflammatory response that is rapidly followed by an anti-inflammatory process, acting in a negative feedback manner (1, 3). These inhibitory mechanisms may become deleterious as nearly all immune functions are compromised. Therefore, they may account for the majority of septic shock related death. Indeed, most nonsurviving patients die after initial resuscitation in a delayed immunosuppressive state (3, 4, 5).

The mechanistic bases for septic shock-induced immunosuppression have not yet been clearly established. It is known that this condition is characterized by profound immunological dysfunction partly due to cell anergy and increased apoptosis of various immune cells (1, 3). Monocytes play a central role in septic shock pathophysiology given that they participate in both the initial inflammatory response and the secondary immunodepression (4). Initially, as components of the innate immune system, they sense microbial products and in response release inflammatory cytokines and initiate adaptative T cell responses due to their capacity of Ag presentation. Subsequently, they exhibit decreased Ag presentation (likely due to a decreased HLA-DR expression), decreased proinflammatory cytokine production, and increased apoptosis (1, 2, 4, 6).

We recently found that the percentage of CD4⁺CD25⁺ regulatory T lymphocytes (Treg)³ is increased in septic shock patients (7, 8). These cells possess potent regulatory properties that are directed at different arms of the immune system (9, 10, 11). Although they have been shown to modulate the innate immune response in various murine models of infectious diseases, their role in human diseases has not yet been established (12). In particular, their potential role in septic shock has never been addressed. As their immunosuppressive properties are not limited to effects on other T cell responses, but also include inhibition of immune pathology mediated by cells of the innate immune system (13), we hypothesized a link between Treg and monocytes. The goal of the current study was thus to examine whether monocyte deactivation might be due to a suppressive effect of Treg during septic shock. We found that CD14 expression is down-regulated in monocytes from septic shock patients and that this effect can be mimicked by incubation of LPS-treated monocytes with conditioned medium from Treg. Treg produce a soluble factor that prevents LPS-induced monocyte survival by a mechanism involving the Fas/FasL pathway, which has previously been linked to sepsis-induced immunosuppression (14). Our findings suggest that Treg may play an important role in the increased level of monocyte apoptosis observed in immunodepressed septic patients.

	Patients, Materials and Methods

Top Abstract Introduction Patients, Materials and Methods Results Discussion Disclosures References

 
Patients

The study group consisted of 33 consecutive patients with septic shock according to the diagnostic criteria of the American College of Chest Physicians/Society of Critical Care Medicine (age, 58 ± 3 years; 9 female, 24 male; mortality, 39%; mean Simplified Acute Physiology Score II on admission, 47, with a range of 15–94). These patients belong to a larger group examined for their percentage of HLA-DR-positive monocytes (results reported elsewhere) (5). Septic shock was defined by an identifiable site of infection, hypotension persisting despite fluid resuscitation, and requiring vasopressor therapy, and evidence of a systemic inflammatory response manifested by at least two of the following criteria: 1) temperature >38°C or <36°C; 2) heart rate >90 beats/min; 3) respiratory rate >20 breaths/min; and 4) white blood cell count >12,000/mm³ or <4,000/mm³. Severity was assessed by the Simplified Acute Physiology Score II. Mortality was defined as death occurring within 28 days after the onset of shock. Cell phenotyping was performed at days 1 and 2 and until day 15 after the onset of shock on residual blood after completing routine follow-up performed in our intensive care unit, in accordance with the human experimentation guidelines for clinical research of our institute. To provide a panel values from healthy donors, we also included 36 individuals from the laboratory staff of our hospital (ages 23–59 years, 27 female, 9 male, without comorbidity) after informed consent was given.

Cell isolation

Heparinized blood was taken from healthy individuals (n = 40) after informed consent was given (age 34 ± 2 years, 21 female, 19 male). Mononuclear cells were isolated from freshly drawn human blood by Ficoll-Paque Plus gradient centrifugation (Amersham). Monocytes were first positively selected using anti-CD14 microbeads (20 µl/10⁷ total cells), incubated for 30 min at 4°C, and positively selected using a MACS column (purity, >90%). CD4⁺CD25⁺ Treg were then purified. Non-CD4⁺ T cells were stained using a biotin Ab mixture (10 µl/10⁷ total cells), incubated for 30 min at 4°C, magnetically labeled with antibiotin microbeads (20 µl/10⁷ total cells), incubated for 30 min at 4°C, and depleted over a MACS column (purity of purified CD4⁺ lymphocytes >90%). CD4⁺CD25⁺ T cells were directly labeled with anti-CD25 microbeads (10 µl/10⁷ total cells), incubated for 30 min at 4°C and positively selected using MACS column according to the manufacturer’s recommendations (Miltenyi Biotec). Purity was assessed by a CD4/CD25/CD45RO staining. T cell subpopulations of CD4⁺ T cells (CD25⁺ or CD25^–) were cultured immediately after isolation.

Cells and culture conditions

Cells were cultured in RPMI 1640 supplemented with 2 nM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich) in 24-well ultralow attachment plates (Corning-Costar). The number of monocytes per well was adjusted to the number of CD4⁺CD25⁺ T lymphocytes (ratio 1:1). LPS was added at a concentration of 10 µg/ml (15). Cells were incubated 16 h at 37°C in a humidified 5% CO₂ atmosphere.

Reagents and flow cytometry

The following Abs were used: FITC- or energy-coupled dye-labeled anti-CD14; PC5-labeled anti-CD45, FITC-labeled anti-CD4, energy-coupled dye-labeled anti-CD45RO, and PE-labeled anti-CD95 (Immunotech); PE- or PC5-labeled anti-CD25 and PE-labeled anti-HLA-DR (BD Biosciences); PE-labeled anti-TLR4 (eBiosciences); and PE-labeled anti-FasL (R&D Systems). FITC-labeled recombinant human annexin V was purchased from Bender MedSystems. 3,3'-Dihexyloxacarbocyanine iodide (DIOC-6) was purchased from Molecular Probes.

The samples were run on an EPICS XL flow cytometer and analyzed using Expo 32 software (Beckman-Coulter).

Unconjugated anti-IL-10, anti-IL-4, anti-TNF- (Diaclone, Besançon, FR), anti-TRAIL (BD Biosciences), and anti-FasL (clone 4H9; Immunotech) Abs were used for neutralization experiments. Anti-FasL Abs neutralizes soluble Fas ligand and membrane-bound Fas ligand cytotoxic activity. Unconjugated anti-Fas Abs (clone 7C11; Immunotech) were used for induction of apoptosis. Recombinant human IL-10 was purchased from R&D Systems. LPS from Escherichia coli O55:B5 and recombinant human Fas-Ligand Set were purchased from Sigma-Aldrich.

CFSE staining and proliferation experiments

Purified CD4⁺CD25^– T cells (5 x 10⁶ cells/ml) were incubated with 10 µM CFSE (Molecular Probes) for 15 min at 37°C in the dark. After a washing, the labeled CD4⁺CD25^– T cells were resuspended in complete culture medium (10% autologous serum) and used as responder cells in proliferation experiments (PHA 2 µg/ml; Sigma-Aldrich). Proliferation was assessed by flow cytometry at day 6 after the onset of culture. Three types of experiments were set up: 1) CFSE-stained CD4⁺CD25^– T cells were cultured with PHA 2 µg/ml; 2) CFSE-stained CD4⁺CD25^– T cells were cultured with PHA and an equal number of nonstained Treg; 3) CFSE-stained CD4⁺CD25^– T cells were cultured with PHA and an equal number of nonstained CD4⁺CD25^– T cells (5 x 10⁵ cells/well).

mRNA microarray hybridization and analysis

To better characterize the cell populations, total RNA extracted from CD4⁺CD25⁺ and CD4⁺CD25^– populations was analyzed using microarrays. CD4⁺CD25⁺ and CD4⁺CD25^– cells were isolated as described above. mRNA extraction was performed on purified cells using RNeasy Mini Kit (Qiagen). Residual DNA contamination was digested using an RNase-free DNase set (Qiagen). We used 200 ng of total RNA to prepare double-stranded cDNA containing the T7 promoter sequence using the two-cycle target labeling assays (Affymetrix). cRNA was synthesized and labeled with biotinylated ribonucleotide by in vitro transcription using the T7 promoter coupled double-stranded cDNA as template and the GeneChip IVT Labeling Kit (Affymetrix). A second round of cDNA synthesis and in vitro transcription reactions was performed as described by the GeneChip Eukaryotic Small Sample Target Labeling Assay Version II (Affymetrix). The fragmented cRNA was hybridized onto HG-U133A oligonucleotide arrays (Affymetrix), containing 22,283 probe sets representing >14,500 well-substantiated human genes. The arrays were washed and stained according to the Affymetrix protocol EukGE-WS2v4 using an Affymetrix fluidic station FS450. The array was scanned with the Agilent G2500A GeneArray Scanner. The perfect match only model RMA (Robust Multichip Average) quantil normalization (16, 17, 18) was conducted using BioConductor package Affy_1.2.30 (Bioconductor project http://www.bioconductor.org). Expression levels were expressed as 2e^{RMA expression level}. Using the NETAFFX web site (www.affymetrix.com), probe sets associated to candidate genes known to be differentially expressed between CD4⁺CD25⁺ and CD4⁺CD25^– populations were selected for expression level analysis.

Transwell experiments

Cells were isolated as described above and cultured in 24-well Transwell plates (0.4 µm; Corning-Costar). LPS was distributed in the lower chamber. CD14 expression was monitored on monocytes on both upper and lower chambers, in the presence or absence of direct cellular contact with an equal number of CD4⁺CD25⁺ or CD4⁺CD25^– T lymphocytes.

Cytokine analysis by ELISA

Supernatants removed after overnight incubation were conserved at –80°C until cytokine were measured using ELISA commercial kits. IL-10 and IL-4 were measured using kits from Biosource. The lower limits of detection were 1 and 2 pg/ml, respectively, according to the manufacturer’s instructions. Soluble FasL measurements were performed using a commercial kit from R&D Systems. The lower limit of detection was 3 pg/ml according to the supplier’s instructions. ELISAs were quantified by OD₄₅₀ on a microplate reader (Dynatech).

Statistics

Data are presented as mean ± SEM. The results are considered as significant at p < 0.05, as determined by the Wilcoxon nonparametric paired test. Due to the sample size, comparisons between groups of patients were made with the nonparametric Mann-Whitney U test without correction on the number of test performed.

	Results

Top Abstract Introduction Patients, Materials and Methods Results Discussion Disclosures References

 
Loss of CD14 expression on monocytes from septic shock patients

During septic shock, monocytes present with decreased response to endotoxin stimulation (1, 6, 19). Surprisingly, little work has been specifically devoted to the study of LPS coreceptors on monocytes. We therefore retrospectively investigated whether the expression of CD14 and TLR4 was altered on monocytes from septic patients during the course of this condition. We monitored CD14 in 33 consecutive patients (between day 1 and day 15 after the onset of shock) in comparison with 36 healthy individuals. In agreement with our hypothesis, in addition with a decreased HLA-DR expression, septic patients displayed during the whole monitoring, a decrease in CD14 expression on monocytes in comparison with healthy individuals (Fig. 1A). When survivors (n = 20) were separated from nonsurvivors (n = 13), the latter had significantly lower CD14 expression (Fig. 1B). CD14 down-regulation was thus present during septic shock and was correlated with severity. In contrast, TLR4 expression by monocytes was the same in septic patients as in healthy control subjects and did not change during the course of this condition (data not shown).

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Loss of CD14 expression on monocytes from septic shock patients. A, The mean fluorescence intensity (MFI) of CD14 was measured by flow cytometry on monocytes from 33 septic patients (between day (D) 1 and day 15 after the onset of shock). As control, values from 36 healthy donors (measured once) were presented (gray zone). B, Survivors (n = 20) were then separated from nonsurvivors (n = 13). Data are presented as mean ± SEM. The non parametric Mann-Whitney tested was used to assess significances between groups without correction on the number of test performed (*, p < 0.05; #, p < 0.005).

To determine whether Treg could have effects on monocytes similar to what we observed in septic shock patients we purified these cells from peripheral blood of healthy individuals. The purity of CD4⁺CD25⁺ lymphocytes was monitored by flow cytometry after each purification (based on CD4/CD25/CD45RO staining, data not shown). mRNA microarray analysis demonstrated that purified CD4⁺CD25⁺ lymphocytes overexpress FOXP3, CD25, and CTLA4 but exhibit decreased expression of CD69 in comparison with their CD25^– counterpart (Fig. 2A). The suppressive capacity of CD4⁺CD25⁺ T lymphocytes on CFSE-stained CD4⁺CD25^– T cell proliferation was also assessed. The addition of an equal number of non-stained CD4⁺CD25⁺ T lymphocytes induced a substantial decrease in CD4⁺CD25^– T cell proliferation in response to PHA (Fig. 2B), whereas the addition of an equal number of nonstained CD4⁺CD25^– T cells had no effect (data not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Purified CD4+CD25+ T cells overexpress CD25 and FOXP3 and possess regulatory activity. CD4+CD25+ and CD4+CD25– T lymphocytes were purified from healthy individuals. A, mRNA extracted from purified CD4+CD25+ or CD25– T cells was analyzed by microarrays. Individual data from four independent mRNA extractions are represented for four different probe sets known to be differentially expressed between CD4+CD25+ and CD4+CD25– populations. , CD4+CD25+ T cell mRNA; •, CD4+CD25– T cell mRNA. B, The regulatory properties of purified CD4+CD25+ T lymphocytes on CD25– T cell proliferation were tested. Purified CD4+CD25– T cells were stained with CFSE (10 µM) and cultured in the presence of PHA (2 µg/ml). Proliferation was measured after 6 days in the absence (a) or in the presence (b) of an equal number of nonstained CD4+CD25+ T cells by flow cytometry. One representative result of five independent experiments is presented.

Treg inhibit LPS-induced retention of CD14 on monocytes

We investigated the effects of Treg on LPS-treated monocytes. Because of the reduced expression of CD14 (Fig. 1) and HLA-DR (5, 20) that we observed on monocytes from septic patients, we were especially interested to determine whether Treg could block the effect of LPS on the expression of these molecules by monocytes. Monocytes were cultured in the presence or absence of LPS (10 µg/ml) with or without an equal number of either Treg or CD4⁺CD25^– T cells. After culture for 16 h, we did not detect any modification of HLA-DR expression in response to either LPS or Treg (data not shown). In the absence of LPS, few monocytes retained CD14 after 16 h (13 ± 3%; Fig. 3, Aa and Ab), whereas in its presence, a distinct population of these cells (44 ± 4%) exhibited near normal CD14 expression (Fig. 3Ac). Addition of Treg to the monocyte cultures completely abrogated LPS-induced retention of CD14 (Fig. 3Ad), whereas CD4⁺CD25^– T cells had no effect (Fig. 3Ae). Overall, CD14 expression was 2.5 times higher in LPS-treated monocytes than in control cells (p < 0.001), and this effect was completely blocked by Treg but not by CD4⁺CD25^– T cells (Fig. 3B).

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Treg inhibits LPS-induced retention of CD14 on monocytes. A, An equal number of purified monocytes and Treg or CD4+CD25– T cells were cultured overnight in the presence of LPS (10 µg/ml). CD14 expression was measured by flow cytometry on monocytes after 16 h of incubation (CD14/CD45 staining). CD45 stainings were used to positively select leukocytes and to eliminate debris. a, monocytes before culture; b, monocytes; c, monocytes + LPS; d, monocytes + LPS + Treg; e, monocytes + LPS + CD4+CD25– T lymphocytes. One representative result of 17 independent experiments is presented. B, The mean fluorescence intensity (MFI) of CD14 expression was measured on monocytes after culture (mean ± SEM; number of monocytes per well = number of lymphocytes (Ly); LPS = 10 µg/ml; n = 17 experiments). The Wilcoxon nonparametric paired test was used to assess differences between culture conditions (**, p < 0,001). Mean (± SEM) percentages of CD14high monocytes were: T0, 100 ± 1%; control, 13 ± 3%; + LPS, 44 ± 4%; + LPS + Treg, 16 ± 2%; + LPS + CD4+CD25–, 33 ± 4%.

In vitro, Treg usually exert their effect by direct cellular contact with responder cells (9). We thus tested the necessity of cell-cell contact for their effect on CD14 expression. Monocytes and Treg were incubated overnight in 24-well Transwell plates, which permit soluble factors to diffuse from one chamber to another but prevent any cell-cell contact. The physical separation of Treg and monocytes did not prevent the ability of Treg to suppress the effect of LPS on CD14 expression (Fig. 4A).

View larger version (12K): [in this window] [in a new window]   FIGURE 4. The effect of Treg is mediated by a soluble factor. A, Treg and monocytes (Mono) were cultured in 24-well Transwell plates (0,4 µm; number of monocytes in culture = number of Treg). LPS was distributed in the lower chamber (10 µg/ml). The mean fluorescence intensity (MFI) of CD14 expression on monocytes was measured by flow cytometry after overnight incubation (CD14/CD45 staining). Results are presented as mean ± SEM of three independent experiments. B, Monocytes were cultured with supernatant of Treg obtained after 4 h of preincubation and with LPS (10 µg/ml; number of Treg preincubated = number of monocytes in culture). The mean fluorescence intensity of CD14 expression on monocytes was measured by flow cytometry after overnight incubation (CD14/CD45 staining). Results are presented as mean ± SEM of six independent experiments. The Wilcoxon nonparametric paired test was used to assess differences between culture conditions (*, p < 0.05).

The soluble factor released by Treg is not identical with IL-4 or IL-10

Human Treg may produce IL-10 (9, 11). Both this cytokine and IL-4 have been implicated in sepsis-induced immunosuppression (2) and might be able to regulate CD14 expression on monocytes (21, 22). These cytokines were measured in conditioned medium from Treg and in coculture supernatants. Under the conditions used, the levels of IL-4 were below the detection limits of our assay. Moreover, anti-IL-4-blocking Abs did not suppress the effect of Treg on LPS-induced CD14 expression (Fig. 5A).

View larger version (16K): [in this window] [in a new window]   FIGURE 5. IL-4 and IL-10 are not responsible for the effect of Treg conditioned medium. A, Treg and monocytes were cultured in the presence of anti-IL-10-blocking Abs (100 ng/ml) or anti-IL-4-blocking Abs (10 µg/ml) and LPS (10 µg/ml, number of monocytes = number of Treg in culture). The mean fluorescence intensity (MFI) of CD14 expression was measured on monocytes by flow cytometry after overnight incubation (CD14/CD45 staining). Results are the mean ± SEM of three independent experiments. B, Monocytes were cultured in the presence of LPS (10 µg/ml) and increasing concentrations of recombinant human IL-10 (0,01 - 0,1 and 1 ng/ml). The mean fluorescence intensity of CD14 was measured on monocytes after overnight incubation (CD14/CD45 staining). Results the mean ± SEM of six independent experiments. The Wilcoxon nonparametric paired test was used to assess significances between culture conditions (*, p < 0.05).

Treg inhibit LPS-induced monocyte survival

There is evidence that monocytes undergoing apoptosis display reduced surface expression of CD14 (23, 24). We therefore hypothesized that Treg act by preventing LPS-induced monocyte survival. To test this hypothesis, we monitored, in addition to CD14, the apoptotic markers annexin V and DIOC-6 in purified monocytes (24). Monocytes cultured for 16 h in the absence of LPS exhibited a profound reduction in CD14 expression coupled to a marked increase in annexin V staining and a decrease in DIOC-6 staining, indicative of increased spontaneous apoptosis (Fig. 6A).

View larger version (27K): [in this window] [in a new window]   FIGURE 6. CD14 regulation on monocytes is related to apoptosis. A, Monocytes were cultured alone. The mean fluorescence intensity (MFI) of CD14 (), annexin V (•) and DIOC-6 () were monitored by flow cytometry every 4 h during overnight culture (CD14-annexin V (A-V) CD45 and DIOC-6/CD45 stainings). Results are presented as mean ± SEM of four independent experiments. B, An equal number of purified monocytes and Treg or CD4+CD25– T cells were cultured overnight in the presence of LPS (10 µg/ml). a, Monocytes before culture; b, monocytes cultured alone; c, monocytes + LPS; d, monocytes + LPS + Treg; e, monocytes + LPS + CD4+CD25– T lymphocytes (Ly). The fluorescence intensity of annexin V was measured on monocytes with CD14high (black histogram) or CD14low expression (open histogram) (CD14-annexin V-CD45 staining). Results are 1 representative result of 14 consecutive independent experiments. C, An equal number of purified monocytes and Treg or CD4+CD25– T cells were cultured in the presence of LPS (10 µg/ml). The percentage of CD14highannexinlow monocytes among total monocytes was monitored after overnight culture. Results are the mean ± SEM of 14 consecutive independent experiments. The Wilcoxon nonparametric paired test was used to test significances between different culture conditions (*, p < 0.05; **, p < 0.01). ECD, energy-coupled dye.

The proapoptotic effect of Treg on monocytes involves the Fas/FasL pathway

To investigate the nature of the soluble proapoptotic factors involved in the effect of Treg, we added blocking Abs directed against FasL, TNF-, and TRAIL to LPS-treated monocyte-Treg cocultures. Neither anti-TNF nor anti-TRAIL Abs had an effect whereas anti-FasL Abs strongly suppressed the blockade of LPS-induced CD14 expression by Treg (Fig. 7A). A similar tendency was observed in coculture experiments in which anti-FasL, added to the bottom chamber with monocytes, reduced the inhibitory effect of Treg, added to the top chamber. However, this effect was less pronounced than in coculture experiments (Fig. 7B), raising the possibility that a small component of the effect of Treg is mediated through a cell-cell contact mechanism.

View larger version (29K): [in this window] [in a new window]   FIGURE 7. Treg effect on monocyte survival involved Fas/FasL pathway. A, Treg and monocytes were cultured in the presence of anti (a)-FasL, anti-TNF- or anti-TRAIL blocking Abs (1 µg/ml) and LPS (10 µg/ml; number of monocytes = number of lymphocytes in culture). The effect of Treg (percentage of inhibition of LPS-induced CD14 increase) was monitored after overnight incubation in the absence (Veh; ) or in the presence () of blocking Abs (CD14/CD45 staining). Results are the mean ± SEM of five independent experiments. The Wilcoxon nonparametric paired test was used to assess significances between culture conditions (*, p < 0.05). B, Treg and monocytes were cultured in 24-well Transwell plates (0.4 µm) in the presence of LPS (10 µg/ml; number of monocytes in culture = number of Treg) and anti-FasL-blocking Abs (1 µg/ml). Monocytes, LPS, and Abs were distributed in the lower chamber. Treg were incubated in the upper chamber. The effect of Treg (percentage of inhibition of LPS-induced CD14 increase) was monitored after overnight incubation in the absence () or in the presence () of blocking Abs (CD14/CD45 staining). Results are the mean ± SEM of three independent experiments. C, The mean fluorescence intensity (MFI) of FasL expression was measured on freshly purified Treg and CD25– T cells by flow cytometry (FasL/CD4/CD25 staining). The results are presented as mean ± SEM of six independent experiments. The Wilcoxon nonparametric paired test was used to assess significances between cells (*, p < 0.05). D, Monocytes were cultured in the presence of LPS (10 µg/ml) and increasing concentrations of agonistic anti-Fas Abs (0.1, 1, 10 µg/ml). The fluorescence intensity of CD14 and annexin V on monocytes were measured by flow cytometry after 16 h of incubation (CD14/annexin V/CD45 staining). One representative result of four independent experiments is presented. E, Monocytes were cultured in the presence of LPS (10 µg/ml) and increasing concentrations of agonistic anti-Fas Abs (0.1, 1, 10 µg/ml). The mean fluorescence intensity of CD14 was measured by flow cytometry after overnight incubation (CD14/annexin V/CD45 staining). Results are the mean ± SEM of four independent experiments. F, Monocytes were cultured in the presence of LPS (10 µg/ml) and increasing concentrations of human recombinant soluble FasL (sFasL; 0.5, 1, 5 ng/ml). The mean fluorescence intensity of CD14 was measured by flow cytometry after overnight incubation (CD14/annexin V/CD45 staining). Results are the mean ± SEM of three independent experiments.

Finally, to determine whether activation of Fas on monocytes could reproduce the effect of Treg, we incubated monocytes in the presence of LPS and increasing concentrations of agonistic anti-Fas Abs. As with Treg conditioned medium, these Abs completely blocked LPS-induced CD14 expression in a dose-dependant manner (Fig. 7, D and E). Identical results were obtained using recombinant human soluble FasL (Fig. 7F).

Collectively, these data support a role for the Fas/FasL proapoptotic pathway in the inhibitory effect of Treg on LPS-induced monocyte survival.

	Discussion

Top Abstract Introduction Patients, Materials and Methods Results Discussion Disclosures References

 
Septic syndromes are still the leading cause of death in intensive care units. In the United States, they develop in 750,000 people annually, of whom >210,000 die (1). Among septic syndromes, septic shock is the most severe, with a mortality ranging from 40 to 50% despite adequate initial treatments. In fact, there is growing recognition that a large number of septic patients rapidly manifest immunosuppression, which may be the principal cause of death, because most of them survive the initial proinflammatory state (1, 2, 4).

An increased percentage of CD4⁺CD25⁺ Treg has been observed in septic patients. Because these cells possess potent regulatory properties on cellular activation (9, 11), they may participate in sepsis-induced immunosuppression. The role of Treg during infectious processes has been studied in mouse models but is still far from being understood in humans (12). Treg can be activated and expand against bacterial, viral, and parasite Ags in vivo. Particularly, Caramalho et al. (15) demonstrated that Treg express various TLRs and that LPS directly activates survival and markedly increases the suppressive activity of Treg through TLR4. Similar results were recently demonstrated using flagellin as a TLR5 ligand (25). Such activated Treg can thus prevent infection-induced immunopathology but may also increase the load of infection and prolong pathogen persistence by suppressing protective immune responses (9, 26). Therefore, their beneficial or deleterious effect is dependent on their relative proportion during infection.

Because the immunosuppressive properties of Treg are not limited to inhibition of T cell responses but also include inhibition of immune pathology mediated by cells of the innate immune system (13), we were prompted to investigate the role of the increased proportion of Treg present during septic shock. We therefore studied the effect of Treg on activation of monocytes, usually considered as key players in septic shock pathophysiology. In a model of LPS-stimulated purified human cells, we describe here two important results: 1) Treg may act on monocytes by inhibiting their LPS-induced survival through a proapoptotic mechanism; 2) this effect is largely mediated by a soluble factor and involves the Fas/FasL pathway.

Relatively little information is available on the interaction of Treg with monocytes. A direct suppressive effect of Treg on APC has been proposed. It has been demonstrated that murine Treg down-regulate the expression of CD80 and CD86 on bone marrow-derived dendritic cells in a cell contact-dependent manner (27). These results were then confirmed in humans (28). Secondly, a recent study by Taams et al. (29) demonstrated that human monocytes incubated with Treg (cell-cell contact) were severely limited in their capacity to induce Ag-specific response and to produce proinflammatory cytokines in response to LPS. However, the mechanisms responsible for these effects were not identified in these models. Thus, Treg may modulate APC functions and thereby make them unable to activate effector T cells.

The present study constitutes the first description of an effect of human Treg on monocyte CD14 expression, which may explain the decreased expression of this molecule on monocytes in septic shock patients. The regulation of CD14 expression on purified human monocytes by cytokines and other factors has received considerable attention (21, 22, 30). CD14 is also regulated on monocytes during their apoptosis (23). In low serum supplementation milieu, it has been shown that monocytes display decreased CD14 expression due to their spontaneous apoptosis (21, 24, 31, 32, 33). This decrease represents an early event during monocyte apoptosis because it precedes annexin V binding, in contrast to HLA expression, which is unchanged in apoptotic monocytes (23). In this context, LPS constitutes a well-known stimulatory factor able to rescue monocytes from apoptosis through c-Flip up-regulation (22, 23, 24, 34).

The major pathway involved in monocyte apoptosis appears to involve Fas/FasL (34, 35, 36, 37). Kiener et al. (37) demonstrated that human monocytes expressed both Fas and FasL and that the autocrine and paracrine interactions of these two molecules are largely responsible for the spontaneous induction of apoptosis that occurs on culture of peripheral monocytes. They also observed that human monocytes contain high levels of intracellular preformed FasL that can be rapidly released (within 30 min) in an active soluble form (36). Finally, Fas may also be required in vivo for regulating circulating monocyte numbers, as mice deficient in the Fas pathway (lpr/lpr) display increased circulating monocytes (35). Taken together, these data are consistent with the proposed role for Fas/FasL in the current study.

Our present data demonstrate that human Treg may participate in Fas/FasL-induced apoptosis by releasing a soluble factor. We observed that Treg suppression of LPS-induced monocyte survival was blocked by anti-FasL Abs and was reproduced by Fas agonist. Moreover, Treg overexpressed FasL in comparison with their CD25^– counterparts. Very few studies have investigated the role of Treg in the induction of apoptosis. It has been recently demonstrated that Treg may use the perforin pathway to kill target cells both in humans (38) and mice (39, 40). Grossman et al. (38) demonstrated that human Treg express granzyme A and display perforin-dependent cytotoxicity. All of these effects necessitated adhesive immunological synapses and were independent of Fas-FasL interactions. In a murine cell line model, Treg were able to lyse Ag-presenting B cells through Fas-FasL interactions in a cell-cell contact-dependent manner. Treg up-regulate FasL expression through which they transduce a death signal, inducing target APC cell apoptosis (41). Our study reports thus on the involvement of human Treg in apoptosis through the release of a soluble factor. The precise mechanisms involved in this effect remain to be further specifically elucidated. Regarding the sensitivity of Treg themselves to apoptosis, contrasting results have been published. Some studies proposed a high sensitivity of Treg to Fas-induced apoptosis (42), whereas others demonstrated the resistance of Treg to apoptosis induced by either Fas (43) or dexamethasone (44). Interestingly, in the context of septic shock, we previously observed that the increased percentage of Treg was due to a reduction in the numbers of CD4⁺CD25^– T cells, whereas the absolute numbers of Treg remained in the normal range (8). To explain this, we proposed that Treg might be resistant to the apoptotic processes occurring after shock. This seems consistent with our current experiments.

In septic shock, apoptotic pathways are largely activated (14, 45). Apoptosis of circulating monocytes has been described in humans (6, 14, 46, 47). Among different mechanisms, the involvement of the Fas/FasL pathway has been well established (48, 49) and seems to be of primary importance given that several studies associated Fas and FasL expression with severity and even mortality in sepsis (50, 51). Furthermore, recent studies using a variety of strategies to inhibit apoptotic processes suggest that blocking programmed cell death is beneficial to sepsis, especially blocking the Fas/FasL pathway (49, 52). Treatments using bcl-2 overexpression (53, 54) and caspase inhibitors have also shown good results in terms of survival (52, 55, 56).

In light of the present study, it seems relevant to propose a link among the increased percentage of Treg, the activated Fas/FasL pathway, and the monocyte apoptosis observed during septic shock. Besides, recent studies observed a participation of Treg in the modulation of the innate immune system during severe inflammatory processes: murine models of septic shock (57) and severe injury (58). It has been also very recently demonstrated that Treg contribute to the development of immune suppression in a mouse burn injury model (59). In summary, we observed that human CD4⁺CD25⁺ Treg inhibit LPS-induced monocyte survival partly through a Fas/FasL mechanism. This constitutes the first description of the involvement of human Treg in an apoptotic process that is largely mediated by the release of a soluble mediator. The identification of the precise mechanism by which Treg induce monocyte apoptosis warrants further investigation. This should give a better understanding of septic shock pathophysiology and may offer new perspectives for the treatment of this deadly disease.

	Acknowledgments

 
We thank H. Thizy, S. Conrozier, and F. Gueyffier (from the Centre d’Investigation Clinique (Clinical Research Centre) of Institut National de la Santé et de la Recherche Médicale and Hospices Civils de Lyon) for logistic support.

	Disclosures

Top Abstract Introduction Patients, Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the Hospices Civils de Lyon (médaille d’or de l’internat) (to F.V.) and the Canadian Institutes of Health Research (to W.S.P.).

² Address correspondence and reprint requests to Dr. G. Monneret, Flow Cytometry Unit-Immunology Laboratory, Hôpital Neurologique, Hospices Civils de Lyon, 59 Boulevard Pinel, 69677 Bron Cedex, France. E-mail address: guillaume.monneret{at}chu-lyon.fr

³ Abbreviations used in this paper: Treg, regulatory T cell; DIOC-6, 3,3'-dihexyloxacarbocyanine iodide.

Received for publication April 12, 2006. Accepted for publication July 14, 2006.

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